Kastle-Meyer test

A dry sample is collected with a swab or filter paper. First a few drops of ethanol, then a few drops of reduced phenolphthalein reagent solution, and, finally, a few drops of hydrogen peroxide are applied to the swab. If the swab turns pink then it is a positive test. This test is nondestructive to the sample, which can be kept and used in further tests at the lab. This test has the same reaction with human blood and animal blood so further testing would be required to determine which one it is.

The phenolphthalein used in this test has been modified from its conventional form, in that it has been reduced by two electrons and is pre-dissolved in alkaline solution. This is typically achieved by boiling an alkaline solution of phenolphthalein with zinc powder, which acts as the reducing agent. Upon reduction, the very intense pink color of the cationic form of phenolphthalein fades to a faint yellow color. It is this form of phenolphthalein that is present in Kastle-Meyer test kits. In order to generate the intense pink color indicative of a positive test, the reduced phenolphthalein must be oxidized back to its normal, colored form.

In the relevant reaction, hydrogen peroxide reacts with the hemoglobin in the blood, with the phenolphthalein not participating in this first process. In its reaction with hydrogen peroxide, the heme center of hemoglobin undergoes the following O-O bond homolysis reaction:

HOOH + Fe3+[heme] → Fe4+=O[heme] + OH· + H+

The products of this reaction are one equivalent each of a high-valent iron-oxo species and hydroxyl radical, either of which can oxidize the reduced phenolphthalein back to its colored form. The amount of acid produced in this reaction is insignificant in comparison to the concentration of base present in the phenolphthalein reagent solution. In addition, this reaction of heme with peroxide is catalytic, making this test very sensitive to small quantities of blood present on the test swab.

This test has some limitations. Namely, the enzymes in some vegetables such as horseradish can cause a false positive test result.[1]

References

^Cox, M. (1991). "A Study of the Sensitivity and Specificity of Four Presumptive Tests for Blood". J. Foren. Sci.36(5).